Methods and compounds for treatment of lymphocyte-related diseases and conditions

ABSTRACT

Methods for treatment of lymphocyte-related diseases and conditions, such as cancer and automimmune diseases, are provided. The methods comprise administration of an effective amount of an oligomer to a patient in need thereof, wherein the oligomer comprises, inter alia, at least one intersubunit linkage having the following structure:wherein R1, L1, X, Y and Z are as defined herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is continuation application of U.S. application Ser.No. 15/579,497, filed Dec. 4, 2017, which is a 35 U.S.C. § 371 filing ofInternational Patent Application No. PCT/US2016/035671, filed Jun. 3,2016, which application claims priority to U.S. Provisional ApplicationSer. No. 62/171,102, filed Jun. 4, 2015, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is SPT_8125USCON_SEQUENCE_LISTING.txt. The textfile is 8.2 KB, was created on Apr. 28, 2021, and is being submittedelectronically via EFS-Web.

BACKGROUND Technical Field

The present invention is generally related to oligonucleotide compounds(oligomers) and methods for their use as antisense compounds, and moreparticularly to methods for use of the oligonucleotide compounds fortreatment of lymphocyte-related diseases and conditions.

Description of the Related Art

Antisense oligonucleotides are useful tools for inhibiting geneexpression and are the subject of ongoing investigation as therapeuticagents. Lymphoid cells, including T lymphocytes and B lymphocytes, areimportant target cells for therapeutic antisense strategies. Lymphocytesare white blood cells that largely mediate the adaptive immune response.Lymphocytes include natural killer (NK) cells, T cells, and B cells. Tcells and B cells are involved in cell-mediated immunity and humoralimmunity, respectively, through their recognition of “non-self” antigensvia cell surface receptors. The antigen receptor of B cells is amembrane bound form of the immunoglobulin that they will secret uponactivation. Upon activation (i.e., binding of the antigen to the B cellreceptor), the B cell differentiates into plasma cells that secreteimmunoglobulins. The antigen receptor of T cells is a membrane boundheterodimeric receptor associated with the proteins of the CD3 complex.Most T lymphocytes have α:β heterodimeric receptors, but some T cellshave γ:δ receptors.

Two simultaneous signals are required to activate a T lymphocyte. Onesignal is provided by a peptide bound to an MHC protein on the surfaceof an antigen presenting cell (APC). The peptide-MHC complex signalsthrough the T cell receptor and its associated proteins. The secondsignal is provided by co-stimulatory molecules on the APC (e.g., CD80and CD86), which are recognized by a co-stimulatory receptor (e.g.,CD28) on the T cell surface. The combination of the two signalsstimulates the T cell to proliferate and begin to differentiate into aneffector T cell (e.g., cytotoxic CD8⁺ T cell, T_(H)1 cell, T_(H)2 cell).Cytotoxic T cells kill infected target cells through the release oflytic granules into antigen-bearing target cells. T_(H)1 cells activatemicrobicidal properties of infected macrophages and induce B cells toproduce opsonizing IgG antibodies. T_(H)2 cells activate naïve B cellsto secrete IgM and induce production of other antibody isotypes.Regulatory T cells (Tregs) are a subpopulation of T cells involved inmaintaining tolerance to self-antigens. Treg function or dysfunction maybe an important factor in numerous pathological settings, includingautoimmunity, allergy, infection, tumor immunity, organ transplantation,and fetal-maternal tolerance.

T cells are frequent therapeutic targets for inhibiting chronicinflammation or cytotoxicity associated with autoimmune diseases. Forexample, defective apoptosis of autoreactive T cells is implicated inthe pathogenesis of multiple sclerosis. Antisense oligonucleotides foran X-linked inhibitor of apoptosis (XIAP) was found to increasesusceptibility of T cells to activation-induced apoptosis in vitro andalleviated experimental autoimmune encephalomyelitis and preventedrelapses in mice (Zehntner et al., 2007, J. Immunol. 179:7553-7560). Inanother example, migration of T lymphocytes into the intestinal mucosaplays a crucial role in the pathogenesis of inflammatory bowel disease(IBD) (e.g., Crohn's disease or ulcerative colitis). Antisenseinhibition of Smad7, an inhibitor of TGF-β1 signaling that isoverexpressed in inflammatory bowel disease mucosa and purified mucosalT cells, enabled IBD cell samples to respond to TGF-β1 and downregulatedproinflammatory cytokines (U.S. Pat. No. 7,700,757; Monteleone et al.,2001, J. Clin. Invest. 108:601-609). In yet another example, synovial Tcells may play a crucial role in rheumatoid arthritis induction andpromotion, in part due to the production of the proinflammatory cytokineIL-17. Antisense targeting of the transcription factor STAT4 in Tlymphocytes suppressed clinical and histopathological signs ofcollagen-induced arthritis in mice (Hildner et al., 2007, J. Immunol.178:3427-3436).

The rejection of allogeneic transplants is largely mediated by T cells.Antisense oligonucleotides have been used to inhibit granzyme expressionin cytotoxic T lymphocytes, which is associated with tissue destructionin transplantation models (Bailey et al., 2004, Eur. J. Immunol.,27:2302-2309).

HIV-infected T lymphocytes, particularly T helper lymphocytes, or CD4⁺ Tlymphocytes, are targets for antisense strategies against HIV(Lisziewicz et al., 1994, Proc. Natl. Acad. Sci. USA 91:7942-7946; Zhanget al., 1995, Clin. Pharmacol. Ther. 58:44-53; Elmen et al., 2004, FEBSLett. 578:285-90; Jakobsen et al., 2007, Retrovirology 4:29-41).

Lymphoid cells are also targeted by antisense oligonucleotides forantitumor therapy. By way of example, human T-cell leukemia virus type 1(HTLV-1) infection is known to cause adult T cell leukemia. Inhibitionof syncytium formation in T-cells was observed in vitro with antisenseoligonucleotides targeting human T-cell leukemia virus type 1 HTLV-1 taxgene (Maeda et al., 1997, Leukemia 11:42-44). In another example,antisense-mediated inhibition of the protooncogene c-myb inhibited DNAsynthesis in T-leukemia cells of most patients (Venturelli et al., 1990Cancer Res. 50:7371-7375). In yet another example, depletion ofregulatory T cells with FOXP3-specific antisense oligonucleotides isbeing investigated as a way to enhance effector T cell response totumors (Morse et al., 2012, Cancer Gene Ther. 19:30-37).

Functional efficacy of such antisense oligonucleotide based therapy islimited by insufficient cellular uptake of the oligonucleotide.Exogenous oligonucleotides are primarily taken up through fluid phaseendocytosis at oligonucleotide concentrations greater than 1 μM andendocytosis mediated by a receptor-like protein for lower concentrations(Beltinger et al., 1995, J. Clin. Invest. 95:1814-1823). Primary cellsare known to incorporate oligonucleotides less efficiently than celllines (Marti et al., 1992, Antisense Res. Dev. 2:27-39). Furthermore,oligonucleotide uptake is heterogenous among different cell types. Amongnormal peripheral blood and bone marrow cells, myeloid cells and B cellspreferably took up more oligonucleotides than T cells (Zhao et al.,1996, Blood 88:1788-1795). Other studies also showed preferential uptakeof oligonucleotides by monocytes and B cells and very low uptake by Tcells (Hartmann et al., 1998, J. Pharmacol. Exp. Ther. 285:920-928;Kronenwett et al., 1998, Blood 91:852-862). In fact, Hartmann et al.demonstrated that only 2% of T cells spontaneously incorporatedFITC-labeled phosphorothioate oligonucleotides as measured byfluorescence intensity. Mitogen activation may increase oligonucleotideuptake (Kreig et al., 1991, Antisense Res. Dev. 1:161-171; Iversen etal., 1992, Antisense Res. Dev. 2:223-233). Cationic lipids have beenused to enhance oligonucleotide uptake into T cells with variableresults (Hartmann et al., supra; Kronenwett et al., supra).

T cells are important targets for inhibition by antisenseoligonucleotides in the treatment of inflammatory, infectious, andneoplastic diseases. There is a need for alternative compositions andmethods for improving uptake of antisense oligonucleotides into T cells.The present disclosure meets this need and further provides otherrelated advantages.

BRIEF SUMMARY

Methods described herein are useful for treatment of various lymphocyte(e.g., T-cell) mediated diseases or conditions. While not wishing to bebound to any particular theory, the present applicants believe thepresence of certain functional groups, such as guanidinyl,alkylguanidinyl and/or alkylaminyl groups, in antisense oligonucleotidesunexpectedly enhances delivery of the antisense oligonucleotides tolymphocytes, such as T-cells. Accordingly, in one embodiment the presentdisclosure provides a method for treatment of a lymphocyte-relateddisease or condition, the method comprising administering an effectiveamount of an oligomer to a patient in need thereof, wherein the oligomercomprises a backbone having a sequence of morpholino ring structuresjoined by intersubunit linkages, the intersubunit linkages joining a3′-end of one morpholino ring structure to a 5′-end of an adjacentmorpholino ring structure, wherein each morpholino ring structure isbound to a base-pairing moiety, such that the oligomer can bind in asequence-specific manner to a target nucleic acid, wherein at least oneof the intersubunit linkages has the following structure (I):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein R¹, L¹, X, Y and Z are as defined herein.

In certain embodiments, the method is for treatment of a T-cell relateddisease or condition, the method comprising contacting activated T-cellswith the above described oligomer.

These and other aspects of the invention will be apparent upon referenceto the following detailed description. To this end, various referencesare set forth herein which describe in more detail certain backgroundinformation, procedures, compounds and/or compositions, and are eachhereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, identical reference numbers identify similar elements.The sizes and relative positions of elements in the figures are notnecessarily drawn to scale and some of these elements are arbitrarilyenlarged and positioned to improve figure legibility. Further, theparticular shapes of the elements as drawn are not intended to conveyany information regarding the actual shape of the particular elements,and have been solely selected for ease of recognition in the figures.

FIG. 1 shows tissue distribution data for a guanidinyl modified oligomerrelative to a peptide conjugated oligomer. AM—abdominal muscle;DP—diaphragm; HT—heart; KD—kidney; L2—liver lobe 2; QC—quadriceps;CD8—CD8 T-cells; Act T reg—activated T-regulatory cells; RestTreg—resting T regulatory cells.

FIG. 2 is a graph showing activity of representative oligomers inactivated T cells.

FIG. 3 presents activity data in B cells.

FIG. 4 compares activity of a guanidinyl modified oligomer in activatedT cells relative to peptide conjugated oligomers.

FIG. 5 illustrates comparative activity of a guanidinyl modifiedoligomer in resting T cells relative to peptide conjugated oligomers.

FIG. 6 presents activity of a guanidinyl modified oligomer in B cellsrelative to peptide conjugated oligomers.

FIG. 7 is a graphical presentation of in vivo data for oligomeractivities in activated T cells.

FIG. 8 shows in vivo activity data in resting T cells.

FIG. 9 presents in vivo activity of various oligomers in T cells.

FIG. 10 provides in vivo activity of various oligomers in macrophages.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments.However, one skilled in the art will understand that the invention maybe practiced without these details. In other instances, well-knownstructures have not been shown or described in detail to avoidunnecessarily obscuring descriptions of the embodiments. Unless thecontext requires otherwise, throughout the specification and claimswhich follow, the word “comprise” and variations thereof, such as,“comprises” and “comprising” are to be construed in an open, inclusivesense, that is, as “including, but not limited to.” Further, headingsprovided herein are for convenience only and do not interpret the scopeor meaning of the claimed invention.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment. Thus, the appearances of the phrases “in one embodiment” or“in an embodiment” in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments. Also, as used in thisspecification and the appended claims, the singular forms “a,” “an,” and“the” include plural referents unless the content clearly dictatesotherwise. It should also be noted that the term “or” is generallyemployed in its sense including “and/or” unless the content clearlydictates otherwise.

The terms below, as used herein, have the following meanings, unlessindicated otherwise:

“Amino” refers to the —NH₂ radical.

“Cyano” or “nitrile” refers to the —CN radical.

“Hydroxy” or “hydroxyl” refers to the —OH radical.

“Imino” refers to the ═NH substituent.

“Guanidinyl” refers to the —NHC(═NH)NH₂ substituent.

“Nitro” refers to the —NO₂ radical.

“Oxo” refers to the ═O substituent.

“Thioxo” refers to the ═S substituent.

“Alkyl” refers to a straight or branched hydrocarbon chain radical whichis saturated or unsaturated (i.e., contains one or more double and/ortriple bonds), having from one to thirty carbon atoms, and which isattached to the rest of the molecule by a single bond. Alkyls comprisingany number of carbon atoms from 1 to 30 are included. An alkylcomprising up to 30 carbon atoms is referred to as a C₁-C₃₀ alkyl,likewise, for example, an alkyl comprising up to 12 carbon atoms is aC₁-C₁₂ alkyl. Alkyls (and other moieties defined herein) comprisingother numbers of carbon atoms are represented similarly. Alkyl groupsinclude, but are not limited to, C₁-C₃₀ alkyl, C₁-C₂₀ alkyl, C₁-C₁₅alkyl, C₁-C₁₀ alkyl, C₁-C₈ alkyl, C₁-C₆ alkyl, C₁-C₄ alkyl, C₁-C₃ alkyl,C₁-C₂ alkyl, C₂-C₈ alkyl, C₃-C₈ alkyl and C₄-C₈ alkyl. Representativealkyl groups include, but are not limited to, methyl, ethyl, n-propyl,1-methylethyl (iso-propyl), n-butyl, i-butyl, s-butyl, n-pentyl,1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl,prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, ethynyl,propynyl, but-2-ynyl, but-3-ynyl, pentynyl, hexynyl, and the like.Unless stated otherwise specifically in the specification, an alkylgroup is optionally substituted as described below.

“Alkylguanidinyl” refers to a radical of the formula —NR_(a)C(═NR_(a))N(R_(a)) 2 where each R_(a) is, independently, H or an alkylradical as defined above, provided at least one R_(a). is an alkylradical Unless stated otherwise specifically in the specification, analkylguanidinyl group is optionally substituted as described below.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group.Alkylenes may be saturated or unsaturated (i.e., contains one or moredouble and/or triple bonds). Representative alkylenes include, but arenot limited to, C₁-C₁₂ alkylene, C₁-C₈ alkylene, C₁-C₆ alkylene, C₁-C₄alkylene, C₁-C₃ alkylene, C₁-C₂ alkylene, C₁ alkylene. Representativealkylene groups include, but are not limited to, methylene, ethylene,propylene, n-butylene, ethenylene, propenylene, n-butenylene,propynylene, n-butynylene, and the like. The alkylene chain is attachedto the rest of the molecule through a single or double bond and to theradical group through a single or double bond. The points of attachmentof the alkylene chain to the rest of the molecule and to the radicalgroup can be through one carbon or any two carbons within the chain.Unless stated otherwise specifically in the specification, an alkylenechain is optionally substituted as described below. For example, incertain embodiments the alkylene is substituted with oxo.

“Aminoalkylene” refers to an alkylene as defined above, wherein thehydrocarbon chain is interrupted by (i.e., includes) at least onenitrogen atom. The nitrogen atom(s) may be at any position in thehydrocarbon chain, including the terminal ends (i.e., the nitrogen atommay link the hydrocarbon chain to the rest of the molecule and/or to theradical group). Unless stated otherwise specifically in thespecification, an aminoalkylene group is optionally substituted asdescribed below.

“Oxyalkylene” refers to an alkylene as defined above, wherein thehydrocarbon chain is interrupted by (i.e., includes) at least one oxygenatom. The oxygen atom(s) may be at any position in the hydrocarbonchain, including the terminal ends (i.e., the oxygen atom may link thehydrocarbon chain to the rest of the molecule and/or to the radicalgroup). Unless stated otherwise specifically in the specification, anoxyalkylene group is optionally substituted as described below.

“Thioalkylene” refers to an alkylene as defined above, wherein thehydrocarbon chain is interrupted by (i.e., includes) at least one sulfuratom. The sulfur atom(s) may be at any position in the hydrocarbonchain, including the terminal ends (i.e., the sulfur atom may link thehydrocarbon chain to the rest of the molecule and/or to the radicalgroup). Unless stated otherwise specifically in the specification, athioalkylene group is optionally substituted as described below.

“Alkylaminyl” refers to a radical of the formula —NHR_(a) or—NR_(a)R_(a) where each R_(a) is, independently, an alkyl radical asdefined above. Unless stated otherwise specifically in thespecification, an alkylamino group is optionally substituted asdescribed below.

“Fused” refers to any ring structure described herein which is fused toan existing ring structure. When the fused ring is a heterocyclyl ringor a heteroaryl ring, any carbon atom on the existing ring structurewhich becomes part of the fused heterocyclyl ring or the fusedheteroaryl ring may be replaced with a nitrogen atom.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodo.

“Heterocyclyl”, “heterocycle” or “heterocyclic ring” refers to a stable3-to 24-membered non-aromatic ring radical comprising 2 to 23 carbonatoms and from one to 8 heteroatoms selected from the group consistingof nitrogen, oxygen, phosphorous and sulfur. Unless stated otherwisespecifically in the specification, the heterocyclyl radical may be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems; and the nitrogen, carbon orsulfur atoms in the heterocyclyl radical may be optionally oxidized; thenitrogen atom may be optionally quaternized; and the heterocyclylradical may be partially or fully saturated. Examples of suchheterocyclyl radicals include, but are not limited to, dioxolanyl,thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl,1,1-dioxo-thiomorpholinyl, 12-crown-4, 15-crown-5, 18-crown-6,21-crown-7, aza-18-crown-6, diaza-18-crown-6, aza-21-crown-7, anddiaza-21-crown-7. Unless stated otherwise specifically in thespecification, a heterocyclyl group may be optionally substituted.

All the above groups may be either substituted or unsubstituted. Theterm “substituted” as used herein means any of the above groups (e.g.,alkyl, alkylguanidinyl, alkylene, aminoalkylene, oxyalkylene,thioalkylene, alkylaminyl and/or heterocyclyl), may be furtherfunctionalized wherein at least one hydrogen atom is replaced by a bondto a non-hydrogen atom substituent. Unless stated specifically in thespecification, a substituted group may include one or more substituentsselected from: amino, oxo, —CO₂H, nitrile, nitro, —CONH₂, hydroxyl,imino, thio, alkyl, alkylene, alkoxy, alkoxyalkyl, alkylcarbonyl,alkyloxycarbonyl, aryl, aralkyl, arylcarbonyl, aryloxycarbonyl,aralkylcarbonyl, aralkyloxycarbonyl, aryloxy, cycloalkyl,cycloalkylalkyl, cycloalkylcarbonyl, cycloalkylalkylcarbonyl,cycloalkyloxycarbonyl, heterocyclyl, heteroaryl, dialkylamines,arylamines, alkylarylamines, diarylamines, N-oxides, imides, andenamines; a silicon atom in groups such as trialkylsilyl groups,dialkylarylsilyl groups, alkyldiarylsilyl groups, triarylsilyl groups,perfluoroalkyl or perfluoroalkoxy, for example, trifluoromethyl ortrifluoromethoxy. “Substituted” also means any of the above groups inwhich one or more hydrogen atoms are replaced by a higher-order bond(e.g., a double- or triple-bond) to a heteroatom such as oxygen in oxo,carbonyl, carboxyl, and ester groups; and nitrogen in groups such asimines, oximes, hydrazones, and nitriles. For example, “substituted”includes any of the above groups in which one or more hydrogen atoms arereplaced with —NR_(g)C(═O)NR_(g)R_(h), —NR_(g)C(═O)OR_(h),—NR_(g)SO₂R_(h), —OC(═O)NR_(g)R_(h), —OR_(g), —SR_(g), —SOR_(g),—SO₂R_(g), —OSO₂R_(g), —SO₂OR_(g), ═NSO₂R_(g), and —SO₂NR_(g)R_(h).“Substituted” also means any of the above groups in which one or morehydrogen atoms are replaced with —C(═O)R_(g), —C(═O)OR_(g),—CH₂SO₂R_(g), —CH₂SO₂NR_(g)R_(h), —SH, —SR_(g) or —SSR_(g). In theforegoing, R_(g) and R_(h) are the same or different and independentlyhydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl,heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl. Inaddition, each of the foregoing substituents may also be optionallysubstituted with one or more of the above substituents. Furthermore, anyof the above groups may be substituted to include one or more internaloxygen or sulfur atoms. For example, an alkyl group may be substitutedwith one or more internal oxygen atoms to form an ether or polyethergroup. Similarly, an alkyl group may be substituted with one or moreinternal sulfur atoms to form a thioether, disulfide, etc.

The terms “antisense oligomer” or “antisense compound” are usedinterchangeably and refer to a sequence of subunits, each having a basecarried on a backbone subunit composed of ribose or other pentose sugaror morpholino group, and where the backbone groups are linked byintersubunit linkages that allow the bases in the compound to hybridizeto a target sequence in a nucleic acid (typically an RNA) byWatson-Crick base pairing, to form a nucleic acid:oligomer heteroduplexwithin the target sequence. The oligomer may have exact sequencecomplementarity to the target sequence or near complementarity. Suchantisense oligomers are designed to block or inhibit translation of themRNA containing the target sequence, and may be said to be “directed to”a sequence with which it hybridizes.

A “morpholino oligomer” or “PMO” refers to a polymeric molecule having abackbone which supports bases capable of hydrogen bonding to typicalpolynucleotides, wherein the polymer lacks a pentose sugar backbonemoiety, and more specifically a ribose backbone linked by phosphodiesterbonds which is typical of nucleotides and nucleosides, but insteadcontains a ring nitrogen with coupling through the ring nitrogen. Anexemplary “morpholino” oligomer comprises morpholino subunit structureslinked together by (thio)phosphoramidate or (thio)phosphorodiamidatelinkages, joining the morpholino nitrogen of one subunit to the 5′exocyclic carbon of an adjacent subunit, each subunit comprising apurine or pyrimidine base-pairing moiety effective to bind, bybase-specific hydrogen bonding, to a base in a polynucleotide.Morpholino oligomers (including antisense oligomers) are detailed, forexample, in U.S. Pat. Nos. 5,698,685; 5,217,866; 5,142,047; 5,034,506;5,166,315; 5,185,444; 5,521,063; 5,506,337 and pending U.S. patentapplication Ser. Nos. 12/271,036; 12/271,040; and PCT publication numberWO/2009/064471 all of which are incorporated herein by reference intheir entirety. Representative PMOs include PMOs wherein theintersubunit linkages are linkage (A1).

A “phosphoramidate” group comprises phosphorus having three attachedoxygen atoms and one attached nitrogen atom, while a“phosphorodiamidate” group comprises phosphorus having two attachedoxygen atoms and two attached nitrogen atoms. In the uncharged or themodified intersubunit linkages of the oligomers described herein andco-pending U.S. Patent Application No. 61/349,783 and Ser. No.11/801,885, one nitrogen is always pendant to the backbone chain. Thesecond nitrogen, in a phosphorodiamidate linkage, is typically the ringnitrogen in a morpholino ring structure.

“Thiophosphoramidate” or “thiophosphorodiamidate” linkages arephosphoramidate or phosphorodiamidate linkages, respectively, whereinone oxygen atom, typically the oxygen pendant to the backbone, isreplaced with sulfur.

“Intersubunit linkage” refers to the linkage connecting two morpholinosubunits, for example structure (I).

“Charged”, “uncharged”, “cationic” and “anionic” as used herein refer tothe predominant state of a chemical moiety at near-neutral pH, e.g.,about 6 to 8. For example, the term may refer to the predominant stateof the chemical moiety at physiological pH, that is, about 7.4.

“Lower alkyl” refers to an alkyl radical of one to six carbon atoms, asexemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl, isoamyl,n-pentyl, and isopentyl. In certain embodiments, a “lower alkyl” grouphas one to four carbon atoms. In other embodiments a “lower alkyl” grouphas one to two carbon atoms; i.e. methyl or ethyl. Analogously, “loweralkenyl” refers to an alkenyl radical of two to six, preferably three orfour, carbon atoms, as exemplified by allyl and butenyl.

A “non-interfering” substituent is one that does not adversely affectthe ability of an antisense oligomer as described herein to bind to itsintended target. Such substituents include small and/or relativelynon-polar groups such as methyl, ethyl, methoxy, ethoxy, or fluoro.

An oligonucleotide or antisense oligomer “specifically hybridizes” to atarget polynucleotide if the oligomer hybridizes to the target underphysiological conditions, with a Tm greater than 37° C., greater than45° C., preferably at least 50° C., and typically 60° C.-80° C. orhigher. The “Tm” of an oligomer is the temperature at which 50%hybridizes to a complementary polynucleotide. Tm is determined understandard conditions in physiological saline, as described, for example,in Miyada et al., Methods Enzymol. 154:94-107 (1987). Such hybridizationmay occur with “near” or “substantial” complementary of the antisenseoligomer to the target sequence, as well as with exact complementarity.

Polynucleotides are described as “complementary” to one another whenhybridization occurs in an antiparallel configuration between twosingle-stranded polynucleotides. Complementarity (the degree that onepolynucleotide is complementary with another) is quantifiable in termsof the proportion of bases in opposing strands that are expected to formhydrogen bonds with each other, according to generally acceptedbase-pairing rules.

A first sequence is an “antisense sequence” with respect to a secondsequence if a polynucleotide whose sequence is the first sequencespecifically binds to, or specifically hybridizes with, the secondpolynucleotide sequence under physiological conditions.

The term “targeting sequence” is the sequence in the oligonucleotideanalog that is complementary (meaning, in addition, substantiallycomplementary) to the target sequence in the RNA genome. The entiresequence, or only a portion, of the analog compound may be complementaryto the target sequence. For example, in some embodiments an analoghaving 20 bases, only 12-14 may be targeting sequences. Typically, thetargeting sequence is formed of contiguous bases in the analog, but mayalternatively be formed of non-contiguous sequences that when placedtogether, e.g., from opposite ends of the analog, constitute sequencethat spans the target sequence.

Target and targeting sequences are described as “complementary” to oneanother when hybridization occurs in an antiparallel configuration. Atargeting sequence may have “near” or “substantial” complementarity tothe target sequence and still function for the purpose of the presentlydescribed methods, that is, still be “complementary.” Preferably, theoligonucleotide analog compounds employed in the presently describedmethods have at most one mismatch with the target sequence out of 10nucleotides, and preferably at most one mismatch out of 20.Alternatively, the antisense oligomers employed have at least 90%sequence homology, and preferably at least 95% sequence homology, withthe exemplary targeting sequences as designated herein. For purposes ofcomplementary binding to an RNA target, and as discussed below, aguanine base may be complementary to either a cytosine or uracil RNAbase.

A “heteroduplex” refers to a duplex between an oligonucleotide analogand the complementary portion of a target RNA. A “nuclease-resistantheteroduplex” refers to a heteroduplex formed by the binding of anantisense oligomer to its complementary target, such that theheteroduplex is substantially resistant to in vivo degradation byintracellular and extracellular nucleases, such as RNAse H, which arecapable of cutting double-stranded RNA/RNA or RNA/DNA complexes.

An agent is “actively taken up by mammalian cells” when the agent canenter the cell by a mechanism other than passive diffusion across thecell membrane. The agent may be transported, for example, by “activetransport”, referring to transport of agents across a mammalian cellmembrane by e.g. an ATP-dependent transport mechanism, or by“facilitated transport”, referring to transport of antisense agentsacross the cell membrane by a transport mechanism that requires bindingof the agent to a transport protein, which then facilitates passage ofthe bound agent across the membrane.

The terms “modulating expression” and/or “antisense activity” refer tothe ability of an antisense oligomer to either enhance or, moretypically, reduce the expression of a given protein, by interfering withthe expression or translation of RNA. In the case of reduced proteinexpression, the antisense oligomer may directly block expression of agiven gene, or contribute to the accelerated breakdown of the RNAtranscribed from that gene. Morpholino oligomers as described herein arebelieved to act via the former (steric blocking) mechanism. Preferredantisense targets for steric blocking oligomers include the ATG startcodon region, splice sites, regions closely adjacent to splice sites,and 5′-untranslated region of mRNA, although other regions have beensuccessfully targeted using morpholino oligomers.

An “effective amount” or “therapeutically effective amount” refers to anamount of antisense oligomer administered to a mammalian subject, eitheras a single dose or as part of a series of doses, which is effective toproduce a desired therapeutic effect, typically by inhibitingtranslation of a selected target nucleic acid sequence.

“Treatment” of an individual (e.g. a mammal, such as a human) or a cellis any type of intervention used in an attempt to alter the naturalcourse of the individual or cell. Treatment includes, but is not limitedto, administration of a pharmaceutical composition, and may be performedeither prophylactically or subsequent to the initiation of a pathologicevent or contact with an etiologic agent.

As used herein, “T cells,” also known as “T lymphocytes,” means a subsetof lymphocytes defined by their development in the thymus and byheterodimeric receptors (T cell receptors) associated with the proteinsof the CD3 complex. Most T lymphocytes have α:β heterodimeric receptors,but some T cells have γ:δ receptors. T cells include several subsets,each with distinct functions: T helper cells (T_(H) cells or CD4⁺ Tcells), cytotoxic T cells (CD8⁺ T cells, Tc cells, or CTLs), memory Tcells, regulatory T cells (T_(reg) cells), and natural killer T cells(NKT cells).

As used herein, an “activated T cells” is a T cell that has beensignaled to proliferate and differentiate into an effector cell. Twosimultaneous signals are required to activate a T cell. One signal isprovided by a peptide bound to an MHC protein on the surface of anantigen presenting cell (APC). The peptide-MHC complex signals throughthe T cell receptor and its associated proteins. The second signal isprovided by co-stimulatory molecules on the APC (e.g., CD80 and CD86),which are recognized by a co-stimulatory receptor (e.g., CD28) on the Tcell surface. The combination of the two signals stimulates the T cellto proliferate and begin to differentiate into an effector cell.

As used herein, “B cells,” also known as “B lymphocytes” means a subsetof lymphocytes having an immunoglobulin molecule as a cell surfacereceptor (B cell receptor). Upon activation by an antigen (i.e., bindingof the antigen to the B cell receptor), a B cell differentiates intoplasma cells producing antibody of the same specificity as its initialreceptor.

A. Methods

As noted above, one embodiment of the present disclosure is directed tomethods for treatment of lymphocyte-related diseases or conditions, forexample T-cell mediated diseases. In general, the methods compriseadministering an oligomer comprising a guanidinyl, alkylguanidinyland/or alkylaminyl moiety to a patient in need thereof. For example, insome embodiments the oligomer is a morpholino oligomer. The guanidinyl,alkylguanidinyl and/or alkylaminyl moiety may be present at any locationwithin the oligomer, and in certain embodiments the guanidinyl,alkylguanidinyl and/or alkylaminyl moiety is bound to the intersubunitlinkage of a morpholino oligomer.

In other embodiments, a method for treatment of a lymphocyte-relateddisease or condition is provided, the method comprising administering aneffective amount of an oligomer to a patient in need thereof, whereinthe oligomer comprises a backbone having a sequence of morpholino ringstructures joined by intersubunit linkages, the intersubunit linkagesjoining a 3′-end of one morpholino ring structure to a 5′-end of anadjacent morpholino ring structure, wherein each morpholino ringstructure is bound to a base-pairing moiety, such that the oligomer canbind in a sequence-specific manner to a target nucleic acid, wherein atleast one of the intersubunit linkages has the following structure (I):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

R¹ is guanidinyl, alkylguanidinyl or alkylaminyl;

L¹ is absent or present, and when present is selected from alkylene,aminoalkylene, oxyalkylene and thioalkylene;

X is, at each occurrence, independently S or O;

Y is, at each occurrence, independently —O— or —NH—; and

Z is an optionally substituted 5, 6 or 7-membered heterocyclic ring.

In certain embodiments of the above, the lymphocyte-related disease orcondition is a T-cell related disease or condition.

Various different oligomers are useful for the described methods. Forexample, morpholino oligomers are employed in various embodiments. Insome of these embodiments, the morpholino ring structures have thefollowing structure (i):

wherein B is, at each occurrence, independently a base-pairing moiety,such as A, C, G, T, I, U and the like.

In various embodiments of the above, Z is an optionally substituted 5 or6-membered heterocyclic ring. For example, in some embodiments Z ispyrrolidinyl, or piperidinyl. In more specific embodiments, Z ispiperidinyl.

In even other embodiments, Z has one of the following structures:

For example, in further embodiments Z has the following structure:

The position of the R¹-L¹ moiety on the Z ring is not particularlylimited and various substitution patterns are expected to result inoligomers having the desired properties (i.e., effective delivery tolymphocytes and treatment of lymphocyte-related diseases and/orconditions). In certain embodiments, Z has the following structure:

In any one of the foregoing embodiments, R¹ is guanidinyl.

In other of any one of the foregoing embodiments, R¹ is alkylguanidinyl.For example, in some embodiments the alkylguanidinyl has the followingstructure:

wherein R′ is C₁-C₆alkyl. In further of these embodiments, R′ is methyl.

In still more of any one of the foregoing embodiments, R¹ isalkylaminyl. For example, in certain of these embodiments thealkylaminyl is —NHR″, where R″ is C₁-C₆alkyl. In further of theseembodiments, R″ is methyl.

In some embodiments, L¹ is absent and R¹ is bound directly to the Zring. In other embodiments, L¹ is selected from an appropriate lengthalkylene, aminoalkylene, oxyalkylene and thioalkylene such that deliveryand or activity of the oligomer is optimized.

In various embodiments, X is O. In other embodiments, Y is —O—. In stillother embodiments, X is O and Y is —O—.

In addition to one or more intersubunit linkages of structure (I), theoligomers typically contain additional intersubunit linkages of variousstructures. The structures of the other intersubunit linkages are notparticularly limited, provided the oligomer comprises at least oneintersubunit linkage of structure (I). In this regard, any of theintersubunit linkages known to one of ordinary skill in the art can beemployed, and such intersubunit linkages will be selected to optimizethe desired properties of oligomer. In some embodiments, at least one ofthe intersubunit linkages has the following structure (II):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein:

R² and R³ are each independently H or C₁-C₆alkyl,

X′ is S or O; and

Y′ is —O— or —NH—.

In certain of the above embodiments, R² and R³ are each methyl. In otherembodiments, X′ is O. In more embodiments, Y′ is —O—.

In some of the foregoing embodiments, each intersubunit linkage in theoligomer is either linkage (I) or linkage (II). That is, certainembodiments are directed to oligomers having intersubunit linkagesselected from structures (I) and (II), provided at least oneintersubunit linkage has structure (I).

In some more embodiments of the above, at least one of the intersubunitlinkages has the following structure:

In some other embodiments of the above, at least one of the intersubunitlinkages has the following structure:

In even other embodiments of the above, at least one of the intersubunitlinkages has the following structure:

In certain embodiments, the lymphocyte-related disease or condition is aT-cell-related disease or condition. In some of these embodiments, theT-cell is an activated T-cell. In other embodiments, the T-cell is a CD4or CD8 cell.

Various lymphocyte-related diseases or conditions can be treated by thedisclosed methods. In various embodiments, the disease or condition iscancer or an autoimmune disease or condition.

In still other embodiments, a method for treatment of a T-cell relateddisease or condition is provided, the method comprising contactingactivated T-cells with an oligomer comprising a backbone having asequence of morpholino ring structures joined by intersubunit linkages,the intersubunit linkages joining a 3′-end of one morpholino ringstructure to a 5′-end of an adjacent morpholino ring structure, whereineach morpholino ring structure is bound to a base-pairing moiety, suchthat the oligomer can bind in a sequence-specific manner to a targetnucleic acid, wherein at least one of the intersubunit linkages has thefollowing structure (I):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein:

R¹ is guanidinyl, alkylguanidinyl or alkylaminyl;

L¹ is absent or present, and when present is selected from alkylene,aminoalkylene, oxyalkylene, oxoalkylene and thioalkylene;

X is, at each occurrence, independently S or O;

Y is, at each occurrence, independently —O— or —NH—; and

Z is an optionally substituted 5, 6 or 7-membered heterocyclic ring.

In various other embodiments of the foregoing, the oligomer andintersubunit linkages are as defined above.

Still other embodiments are directed to use of an oligomer comprising abackbone having a sequence of morpholino ring structures joined byintersubunit linkages, the intersubunit linkages joining a 3′-end of onemorpholino ring structure to a 5′-end of an adjacent morpholino ringstructure, wherein each morpholino ring structure is bound to abase-pairing moiety, such that the oligomer can bind in asequence-specific manner to a target nucleic acid, wherein at least oneof the intersubunit linkages has the following structure (I):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein:

R₁ is guanidinyl, alkylguanidinyl or alkylaminyl;

L¹ is absent or present, and when present is selected from alkylene,aminoalkylene, oxyalkylene, oxoalkylene and thioalkylene;

X is, at each occurrence, independently S or O;

Y is, at each occurrence, independently —O— or —NH—; and

Z is an optionally substituted 5, 6 or 7-membered heterocyclic ring,

for preparation of a pharmaceutical composition for treatment of alymphocyte-related disease or condition.

In various other embodiments of the foregoing, the oligomer andintersubunit linkages are as defined above.

Another embodiment provides a method for improving delivery of anoligonucleotide to lymphocytes, such as T cell. The method comprisesmodifying the oligonucleotide to contain an intersubunit linkage havingstructure (I).

B. Properties of the Oligomers

As noted above, the present disclosure is directed to methods fortreatment of lymphocyte-related diseases or conditions by administrationof oligomers comprising various intersubunit linkage modifications, suchas linkages comprising guanidinyl, alkylguanidinyl and/or alkylaminylmoieties. In certain embodiments, the oligomer comprises a backbonecomprising a sequence of morpholino ring structures joined byintersubunit linkages, the intersubunit linkages joining a 3′-end of onemorpholino ring structure to a 5′-end of an adjacent morpholino ringstructure, wherein each morpholino ring structure is bound to abase-pairing moiety, such that the oligomer can bind in asequence-specific manner to a target nucleic acid. The morpholino ringstructures may have the following structure (i):

wherein B is, at each occurrence, independently a base-pairing moiety.

Each morpholino ring structure supports a base pairing moiety (B), toform a sequence of base pairing moieties which is typically designed tohybridize to a selected antisense target in a cell or in a subject beingtreated. The base pairing moiety may be a purine or pyrimidine found innative DNA or RNA (A, G, C, T, or U) or an analog, such as hypoxanthine(the base component of the nucleoside inosine) or 5-methyl cytosine.Analog bases that confer improved binding affinity to the oligomer canalso be utilized. Exemplary analogs in this regard includeC5-propynyl-modified pyrimidines, 9-(aminoethoxy)phenoxazine (G-clamp)and the like.

The oligomer may be modified, in accordance with an aspect of theinvention, to include one or more linkages comprising guanidinyl,alkylguanidinyl and/or alkylaminyl moieties, e.g. up to about 1 perevery 2-5 linkages, typically 3-5 per every 10 linkages. Certainembodiments also include one or more linkages comprising guanidinyl,alkylguanidinyl and/or alkylaminyl moieties.

Oligomers for use in antisense applications generally range in lengthfrom about 10 to about 40 subunits, more preferably about 15 to 25subunits. For example, in some embodiments, an oligomer of the inventionhaving 19-20 subunits, a useful length for an antisense oligomer, mayideally have one to seven, e.g. four to six, or three to five linkagescomprising guanidinyl, alkylguanidinyl and/or alkylaminyl moieties.

The morpholino subunits may also be linked by non-phosphorus-basedintersubunit linkages.

Other oligonucleotide analog linkages which are uncharged in theirunmodified state but which could also bear a pendant amine substituentcan also be used. For example, in certain embodiments a 5′nitrogen atomon a morpholino ring could be employed in a sulfamide linkage (or a urealinkage, where phosphorus is replaced with carbon or sulfur,respectively).

In some embodiments for antisense applications, the oligomer may be 100%complementary to the nucleic acid target sequence, or it may includemismatches, e.g., to accommodate variants, as long as a heteroduplexformed between the oligomer and nucleic acid target sequence issufficiently stable to withstand the action of cellular nucleases andother modes of degradation which may occur in vivo. Mismatches, ifpresent, are less destabilizing toward the end regions of the hybridduplex than in the middle. The number of mismatches allowed will dependon the length of the oligomer, the percentage of G:C base pairs in theduplex, and the position of the mismatch(es) in the duplex, according towell understood principles of duplex stability. Although such anantisense oligomer is not necessarily 100% complementary to the nucleicacid target sequence, it is effective to stably and specifically bind tothe target sequence, such that a biological activity of the nucleic acidtarget, e.g., expression of encoded protein(s), is modulated.

The stability of the duplex formed between an oligomer and the targetsequence is a function of the binding T_(m) and the susceptibility ofthe duplex to cellular enzymatic cleavage. The T_(m) of an antisensecompound with respect to complementary-sequence RNA may be measured byconventional methods, such as those described by Hames et al., NucleicAcid Hybridization, IRL Press, 1985, pp. 107-108 or as described inMiyada C. G. and Wallace R. B., 1987, Oligonucleotide hybridizationtechniques, Methods Enzymol. Vol. 154 pp. 94-107.

In some embodiments, each antisense oligomer has a binding T_(m), withrespect to a complementary-sequence RNA, of greater than human bodytemperature or in other embodiments greater than 50° C. In otherembodiments T_(m)'s are in the range 60-80° C. or greater. According towell known principles, the T_(m) of an oligomer compound, with respectto a complementary-based RNA hybrid, can be increased by increasing theratio of C:G paired bases in the duplex, and/or by increasing the length(in base pairs) of the heteroduplex. At the same time, for purposes ofoptimizing cellular uptake, it may be advantageous to limit the size ofthe oligomer. For this reason, compounds that show high T_(m) (50° C. orgreater) at a length of 20 bases or less are generally preferred overthose requiring greater than 20 bases for high T_(m) values. For someapplications, longer oligomers, for example longer than 20 bases mayhave certain advantages. For example, in certain embodiments longeroligomers may find particular utility for use in exon skipping or splicemodulation.

The targeting sequence bases may be normal DNA bases or analoguesthereof, e.g., uracil and inosine that are capable of Watson-Crick basepairing to target-sequence RNA bases.

The oligomers may also incorporate guanine bases in place of adeninewhen the target nucleotide is a uracil residue. This is useful when thetarget sequence varies across different target alleles or viral speciesand the variation at any given nucleotide residue is either cytosine oruracil. By utilizing guanine in the targeting oligomer at the positionof variability, the well-known ability of guanine to base pair withuracil (termed C/U:G base pairing) can be exploited. By incorporatingguanine at these locations, a single oligomer can effectively target awider range of RNA target variability.

The compounds (e.g., oligomers, morpholino subunits, intersubunitlinkages, etc.) may exist in different isomeric forms, for examplestructural isomers (e.g., tautomers). With regard to stereoisomers, thecompounds may have chiral centers and may occur as racemates,enantiomerically enriched mixtures, individual enantiomers, mixture ordiastereomers or individual diastereomers. All such isomeric forms areincluded within the present invention, including mixtures thereof. Thecompounds may also possess axial chirality which may result inatropisomers. Furthermore, some of the crystalline forms of thecompounds may exist as polymorphs, which are included in the presentinvention. In addition, some of the compounds may also form solvateswith water or other organic solvents. Such solvates are similarlyincluded within the scope of this invention.

The oligomers described herein may be used in methods of inhibitingproduction of a protein or replication of a virus. Accordingly, in oneembodiment a nucleic acid encoding such a protein is exposed to anoligomer as disclosed herein. In further embodiments of the foregoing,the antisense oligomer comprises one or more intersubunit linkagescomprising a guanidinyl, alkylguanidinyl and/or alkylaminyl moiety, asdisclosed herein, and the base pairing moieties B form a sequenceeffective to hybridize to a portion of the nucleic acid at a locationeffective to inhibit production of the protein. In one embodiment, thelocation is an ATG start codon region of an mRNA, a splice site of apre-mRNA, or a viral target sequence as described below.

In one embodiment, the oligomer has a T_(m) with respect to binding tothe target sequence of greater than about 50° C., and it is taken up bymammalian cells or bacterial cells.

The preparation and properties of morpholino oligomers is described inmore detail below and in U.S. Pat. No. 5,185,444 and WO/2009/064471,each of which is hereby incorporated by reference in their entirety.

C. Formulation and Administration of the Oligomers

The present disclosure also provides for formulation and delivery of thedisclosed oligomers (e.g., for treatment of lymphocyte-related diseasesand/or conditions). Accordingly, in one embodiment the presentdisclosure is directed to a composition comprising an oligomer asdisclosed herein and a pharmaceutically acceptable vehicle. Otherembodiments are directed to use of such a composition for treatment of alymphocyte-related disease and/or condition.

Effective delivery of the antisense oligomer to the target nucleic acidis an important aspect of treatment. Routes of antisense oligomerdelivery include, but are not limited to, various systemic routes,including oral and parenteral routes, e.g., intravenous, subcutaneous,intraperitoneal, and intramuscular, as well as inhalation, transdermaland topical delivery. The appropriate route may be determined by one ofskill in the art, as appropriate to the condition of the subject undertreatment. For example, the oligomer may be delivered directly to thebloodstream.

The antisense oligomer may be administered in any convenient vehiclewhich is physiologically and/or pharmaceutically acceptable. Such acomposition may include any of a variety of standard pharmaceuticallyacceptable carriers employed by those of ordinary skill in the art.Examples include, but are not limited to, saline, phosphate bufferedsaline (PBS), water, aqueous ethanol, emulsions, such as oil/wateremulsions or triglyceride emulsions, tablets and capsules. The choice ofsuitable physiologically acceptable carrier will vary depending upon thechosen mode of administration.

The compounds (e.g., oligomers) of the present invention may generallybe utilized as the free acid or free base. Alternatively, the compoundsof this invention may be used in the form of acid or base additionsalts. Acid addition salts of the free amino compounds of the presentinvention may be prepared by methods well known in the art, and may beformed from organic and inorganic acids. Suitable organic acids includemaleic, fumaric, benzoic, ascorbic, succinic, methanesulfonic, acetic,trifluoroacetic, oxalic, propionic, tartaric, salicylic, citric,gluconic, lactic, mandelic, cinnamic, aspartic, stearic, palmitic,glycolic, glutamic, and benzenesulfonic acids. Suitable inorganic acidsinclude hydrochloric, hydrobromic, sulfuric, phosphoric, and nitricacids. Base addition salts included those salts that form with thecarboxylate anion and include salts formed with organic and inorganiccations such as those chosen from the alkali and alkaline earth metals(for example, lithium, sodium, potassium, magnesium, barium andcalcium), as well as the ammonium ion and substituted derivativesthereof (for example, dibenzylammonium, benzylammonium,2-hydroxyethylammonium, and the like). Thus, the term “pharmaceuticallyacceptable salt” of structure (I) is intended to encompass any and allacceptable salt forms.

In addition, prodrugs are also included within the context of thisinvention. Prodrugs are any covalently bonded carriers that release acompound of structure (I) in vivo when such prodrug is administered to apatient. Prodrugs are generally prepared by modifying functional groupsof the active moiety in a way such that the modification is cleaved,either by routine manipulation or in vivo, yielding the parent compound.Prodrugs include, for example, compounds of this invention whereinhydroxy, amine or sulfhydryl groups are bonded to any group that, whenadministered to a patient, cleaves to form the hydroxy, amine orsulfhydryl groups. Thus, representative examples of prodrugs include(but are not limited to) acetate, formate and benzoate derivatives ofalcohol and amine functional groups of the compounds of structure (I).Further, in the case of a carboxylic acid (—COOH), esters may beemployed, such as methyl esters, ethyl esters, and the like.

In some instances, liposomes may be employed to facilitate uptake of theantisense oligonucleotide into cells. (See, e.g., Williams, S. A.,Leukemia 10(12):1980-1989, 1996; Lappalainen et al., Antiviral Res.23:119, 1994; Uhlmann et al., antisense oligonucleotides: a newtherapeutic principle, Chemical Reviews, Volume 90, No. 4, pages544-584, 1990; Gregoriadis, G., Chapter 14, Liposomes, Drug Carriers inBiology and Medicine, pp. 287-341, Academic Press, 1979). Hydrogels mayalso be used as vehicles for antisense oligomer administration, forexample, as described in WO 93/01286. Alternatively, theoligonucleotides may be administered in microspheres or microparticles.(See, e.g., Wu, G. Y. and Wu, C. H., J. Biol. Chem. 262:4429-4432,1987). Alternatively, the use of gas-filled microbubbles complexed withthe antisense oligomers can enhance delivery to target tissues, asdescribed in U.S. Pat. No. 6,245,747. Sustained release compositions mayalso be used. These may include semipermeable polymeric matrices in theform of shaped articles such as films or microcapsules.

In one embodiment, antisense inhibition is effective in treating alymphocyte-related disease or conditions, by contacting lymphocytes,such as T cells, with an antisense agent described herein. The antisenseagent is administered to a mammalian subject, e.g., human or domesticanimal, in a suitable pharmaceutical carrier.

In one aspect of the method, the subject is a human subject, e.g., apatient diagnosed as having a lymphocyte-related disease or condition.The condition of a patient may also dictate prophylactic administrationof an antisense oligomer of the invention, e.g. in the case of a patientwho (1) is immunocompromised; (2) is a burn victim; (3) has anindwelling catheter; or (4) is about to undergo or has recentlyundergone surgery. In one preferred embodiment, the oligomer is aphosphorodiamidate morpholino oligomer, contained in a pharmaceuticallyacceptable carrier, and is delivered orally. In another preferredembodiment, the oligomer is a phosphorodiamidate morpholino oligomer,contained in a pharmaceutically acceptable carrier, and is deliveredintravenously (i.v.).

In another application of the method, the subject is a livestock animal,e.g., a chicken, turkey, pig, cow, horse or goat, etc, and the treatmentis either prophylactic or therapeutic.

In one embodiment, the antisense compound is administered in an amountand manner effective to result in a peak blood concentration of at least200-400 nM antisense oligomer. Typically, one or more doses of antisenseoligomer are administered, generally at regular intervals, for a periodof about one to two weeks. Preferred doses for oral administration arefrom about 1-1000 mg oligomer per 70 kg. In some cases, doses of greaterthan 1000 mg oligomer/patient may be necessary. For i.v. administration,preferred doses are from about 0.5 mg to 1000 mg oligomer per 70 kg. Theantisense oligomer may be administered at regular intervals for a shorttime period, e.g., daily for two weeks or less. However, in some casesthe oligomer is administered intermittently over a longer period oftime. Administration may be followed by, or concurrent with,administration of an antibiotic or other therapeutic treatment. Thetreatment regimen may be adjusted (dose, frequency, route, etc.) asindicated, based on the results of immunoassays, other biochemical testsand physiological examination of the subject under treatment.

An effective in vivo treatment regimen using the antisenseoligonucleotides of the invention may vary according to the duration,dose, frequency and route of administration, as well as the condition ofthe subject under treatment. Accordingly, such in vivo therapy willoften require monitoring by tests appropriate to the particular type ofdisease or condition under treatment, and corresponding adjustments inthe dose or treatment regimen, in order to achieve an optimaltherapeutic outcome. Treatment may be monitored, e.g., by generalindicators of disease, such as complete blood count (CBC), nucleic aciddetection methods, immunodiagnostic tests, or detection of heteroduplex.

The efficacy of an in vivo administered antisense oligomer of theinvention in treating lymphocyte-related diseases and/or conditions maybe determined from biological samples (tissue, blood, urine etc.) takenfrom a subject prior to, during and subsequent to administration of theantisense oligomer. Assays of such samples include (1) monitoring thepresence or absence of heteroduplex formation with target and non-targetsequences, using procedures known to those skilled in the art, e.g., anelectrophoretic gel mobility assay.

D. Preparation of the Oligomers

The morpholino subunits, the modified intersubunit linkages andoligomers comprising the same can be prepared as described in theexamples and in U.S. Pat. Nos. 5,185,444; 7,943,762; 8,076,476; and8,299,206 and co-pending U.S. application Ser. No. 13/118,298, which arehereby incorporated by reference in their entirety. The morpholinosubunits can be prepared according to the following general ReactionScheme I.

Referring to Reaction Scheme 1, wherein B represents a base pairingmoiety and PG represents a protecting group, the morpholino subunits maybe prepared from the corresponding ribinucleoside (1) as shown. Themorpholino subunit (2) may be optionally protected by reaction with asuitable protecting group precursor, for example trityl chloride. The 3′protecting group is generally removed during solid-state oligomersynthesis as described in more detail below. The base pairing moiety maybe suitably protected for solid-phase oligomer synthesis. Suitableprotecting groups include benzoyl for adenine and cytosine, phenylacetylfor guanine, and pivaloyloxymethyl for hypoxanthine (I). Thepivaloyloxymethyl group can be introduced onto the N1 position of thehypoxanthine heterocyclic base. Although an unprotected hypoxanthinesubunit, may be employed, yields in activation reactions are farsuperior when the base is protected. Other suitable protecting groupsinclude those disclosed in U.S. Pat. No. 8,076,476, which is herebyincorporated by reference in its entirety.

Reaction of 3 with the activated phosphorous compound 4a or 4b resultsin morpholino subunits having the desired linkage moiety (5a or 5b). Itshould be noted that the R¹ and/or L¹ moieties may also be installed onthe heterocyclic ring Z after formation of the P—C bond or even afterthe subunit has been incorporated into an oligomer.

Compounds of structure 4a or 4b can be prepared using any number ofmethods known to those of skill in the art, including those described inthe examples. Coupling with the morpholino moiety then proceeds asoutlined above.

Compounds of structure 5a or 5b can be used in solid-phase automatedoligomer synthesis for preparation of oligomers comprising theintersubunit linkages. Such methods are well known in the art. Briefly,a compound of structure 5a or 5b may be modified at the 5′ end tocontain a linker to a solid support. Once supported, the protectinggroup of 5a or 5b (e.g., trityl) is removed and the free amine isreacted with an activated phosphorous moiety of a second compound ofstructure 5 (or analogue thereof). This sequence is repeated until thedesired length oligo is obtained. The protecting group in the terminal5′ end may either be removed or left on if a 5′-modification is desired.The oligo can be removed from the solid support using any number ofmethods, or example treatment with a base to cleave the linkage to thesolid support.

The preparation of modified morpholino subunits and morpholino oligomersare described in more detail in the Examples. The morpholino oligomerscontaining any number of modified linkages may be prepared using methodsdescribed herein, methods known in the art and/or described by referenceherein.

E. Antisense Targets of the Oligomers

The present applicants have unexpectedly discovered that the describedoligomers (e.g., oligomers comprising gaunidinyl, alkylguanidinyl and/oralkylaminyl substituents on one or more intersubunit linkage) are highlyeffective for delivery to lymphocytes, such as T cells. Accordingly, thedescribed methods can be employed for treatment of any number oflymphocyte related diseases and/or conditions. In this regard, one ofordinary skill in the art will recognize the various diseases and/orconditions treatable with the oligomers and the associated target (e.g.,gene sequence).

In general, the oligomers comprise a sequence targeted against a geneassociated with a lymphocyte related disease or condition. Typicaltargeting sites within such genes include, but are not limited to startcodons or splice junction sites, for example an ATG start codon regionof an mRNA or a splice site of a pre-mRNA. Accordingly, certainoligomers of the invention comprise or consist of base sequences whichspecifically hybridize to such gene targets. In some embodiments, theoligomers have at least 90% sequence homology or at least 95% sequencehomology, with the targeting site. Exemplary targets are provided inTable 1. Other targets are derivable by one of ordinary skill in theart.

TABLE 1 Exemplary Antisense Targets Cell or Protein Target FunctionIndication T- FoxP3 T regulatory cell transcription Cancer Regulatoryfactor Cells PP1 Inhibitor of FoxP3 Autoimmune Disease Foxo1Instrumental transcription factor Cancer for T regulatory cells PD-1PD-1 Downregulates CD8 T cell Cancer activation TRAF 1 Inhibitor of PD-1expression Autoimmune Disease CTLA-4 CTLA-4 Induces ligand independentAutoimmune SA2 CTLA-4 isoform Disease CTLA-4 Induces soluble CTLA-4Cancer SA3 isoform Th17 IL-17RC Combines with IL-17RA to find AutoimmuneIL-17A and IL-17F Disease IL-17RA Combines with IL-17RA to findAutoimmune IL-17A and IL-17F Disease RORγ Th17 transcription factorAutoimmune Disease IL-22 Promotes the homestasis of Autoimmune epitheliaand is involved in early Disease host defense against microbial(Psoriasis & Brain pathogens Inflammation)

This description is not meant to limit the invention in any way butserves to exemplify the range of human and animal disease conditionsthat can be addressed using oligomers comprising the modifiedintersubunit linkages described herein.

EXAMPLES

Unless otherwise noted, all chemicals were obtained fromSigma-Aldrich-Fluka. Benzoyl adenosine, benzoyl cytidine, andphenylacetyl guanosine were obtained from Carbosynth Limited, UK.

Synthesis of PMO and PMO containing further linkage modifications asdescribed herein was done using methods known in the art and describedin pending U.S. application Ser. Nos. 12/271,036 and 12/271,040 and PCTpublication number WO/2009/064471, which are hereby incorporated byreference in their entirety.

PMO with a 3′ trityl modification are synthesized essentially asdescribed in PCT publication number WO/2009/064471 with the exceptionthat the detritylation step is omitted.

Example 1 tert-butyl(2,2,2-trifluoroacetamido)piperidine-1-carboxylate

To a suspension of tert-butyl 4-aminopiperidine-1-carboxylate (48.7 g,0.243 mol) and DIPEA (130 mL, 0.749 mol) in DCM (250 mL) was added ethyltrifluoroacetate (35.6 mL, 0.300 mol) dropwise while stirring. After 20hours, the solution was washed with citric acid solution (200 mL×3, 10%w/v aq) and sodium bicarbonate solution (200 mL×3, conc aq), dried(MgSO₄), and filtered through silica (24 g). The silica was washed withDCM and the combined eluant was partially concentrated (100 mL), andused directly in the next step. APCI/MS calcd. for C₁₂H₁₉F₃N₂O₃ 296.1,found m/z=294.9 (M−1).

Example 2 2,2,2-trifluoro-N-(piperidin-4-yl)acetamide Hydrochloride

To a stirred DCM solution of the title compound of Example 1 (100 mL)was added dropwise a solution of hydrogen chloride (250 mL, 1.0 mol) in1,4-dioxane (4 M). Stirring was continued for 6 hours, then thesuspension was filtered, and the solid washed with diethyl ether (500mL) to afford the title compound (54.2 g, 96% yield) as a white solid.APCI/MS calcd. for C₇H₁₁F₃N₂O 196.1, found m/z=196.9 (M+1).

Example 3 (4-(2,2,2-trifluoroacetamido)piperidin-1-yl)phosphonicDichloride

To a cooled (ice/water bath) suspension of the title compound of Example2 (54.2 g, 0.233 mol) in DCM (250 mL) was added dropwise phosphorusoxychloride (23.9 mL, 0.256 mol) and DIPEA (121.7 mL, 0.699 mol) andstirred. After 15 minutes, the bath was removed and with continuedstirring the mixture allowed to warm to ambient temperature. After 1hour, the mixture was partially concentrated (100 mL), the suspensionfiltered, and the solid washed with diethyl ether to afford the titlecompound (43.8 g, 60% yield) as a white solid. The eluant was partiallyconcentrated (100 mL), the resulting suspension filtered, and the solidwashed with diethyl ether to afford additional title compound (6.5 g, 9%yield). ESI/MS calcd. for 1-(4-nitrophenyl)piperazine derivativeC₁₇H₂₂ClF₃N₅O₄P 483.1, found m/z=482.1 (M−1).

Example 4((2S,6S)-6-((R)-5-methyl-2,6-dioxo-1,2,3,6-tetrahydropyridin-3-yl)-4-tritylmorpholin-2-yl)methyl(4-(2,2,2-trifluoroacetamido)piperidin-1-yl)phosphonochloridate

To a stirred, cooled (ice/water bath) solution of the title compound ofExample 3 (29.2 g, 93.3 mmol) in DCM (100 mL) was added dropwise over 10minutes a DCM solution (100 mL) of Mo(Tr)T # (22.6 g, 46.7 mmol),2,6-Lutidine (21.7 mL, 187 mmol), and 4-(dimethylamino)pyridine (1.14 g,9.33 mmol). The bath was allowed to warm to ambient temperature. After15 hours, the solution was washed with a citric acid solution (200 mL×3,10% w/v aq), dried (MgSO₄), concentrated, and the crude oil was loadeddirectly onto column. Chromatography [SiO₂ column (120 g), hexanes/EtOAceluant (gradient 1:1 to 0:1), repeated ×3] fractions were concentratedto provide the title compound (27.2 g, 77% yield) as a white solid.ESI/MS calcd. for the 1-(4-nitrophenyl)piperazine derivativeC₄₆H₅₀F₃N₈O₈P 930.3, found m/z=929.5 (M−1).

Example 5((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl(4-(2,2,2-trifluoroacetamido)piperidin-1-yl)phosphonochloridate

The title compound was synthesized in a manner analogous to thatdescribed in Example 4 to afford the title compound (15.4 g, 66% yield)as a white solid. ESI/MS calcd. for 1-(4-nitrophenyl)piperazinederivative C₅₃H₅₃F₃N₁₁O₇P 1043.4, found m/z=1042.5 (M−1).

Example 6 Global Guanidinylation of Oligomers

An appropriate amount of an oligomer containing an aminopiperidinelinkage (or methylated analogue thereof) prepared as described above (25mg, 2.8 μmol) was weighed into a vial (6 ml).1H-Pyrozole-1-carboxamidine chloride (15 mg, 102 μmol) and potassiumcarbonate (20 mg, 0.15 mmol) were added to the vial. Water was added(500 ul), and the reaction mixture was stirred at room temperatureovernight (about 18 hours). Reaction completion was determined by MALDI.

Once complete, the reaction was diluted with 1% ammonia in water (10 ml)and loaded on to an SPE column (2 cm). The vial was rinsed with 1%ammonia solution (2×2 ml), and the SPE column was washed with 1% ammoniain water (3×6 ml). Product was eluted with 45% acetonitrile in 1%ammonia in water (6 ml). Fractions containing oligomer were identifiedby UV optical density measurement. Product was isolated bylyophilization. Purity and identity were determined by MALDI and HPLC(C-18 and/or SAX).

Example 7 Preparation of Morpholino Oligomers

Preparation of trityl piperazine phenyl carbamate 35 (see FIG. 3): To acooled suspension of compound 11 in dichloromethane (6 mL/g 11) wasadded a solution of potassium carbonate (3.2 eq) in water (4 mL/gpotassium carbonate). To this two-phase mixture was slowly added asolution of phenyl chloroformate (1.03 eq) in dichloromethane (2 g/gphenyl chloroformate). The reaction mixture was warmed to 20° C. Uponreaction completion (1-2 hr), the layers were separated. The organiclayer was washed with water, and dried over anhydrous potassiumcarbonate. The product 35 was isolated by crystallization fromacetonitrile. Yield=80%

Preparation of carbamate alcohol 36: Sodium hydride (1.2 eq) wassuspended in 1-methyl-2-pyrrolidinone (32 mL/g sodium hydride). To thissuspension were added triethylene glycol (10.0 eq) and compound 35 (1.0eq). The resulting slurry was heated to 95° C. Upon reaction completion(1-2 hr), the mixture was cooled to 20° C. To this mixture was added 30%dichloromethane/methyl tert-butyl ether (v:v) and water. Theproduct-containing organic layer was washed successively with aqueousNaOH, aqueous succinic acid, and saturated aqueous sodium chloride. Theproduct 36 was isolated by crystallization from dichloromethane/methyltert-butyl ether/heptane. Yield=90%.

Preparation of Tail acid 37: To a solution of compound 36 intetrahydrofuran (7 mL/g 36) was added succinic anhydride (2.0 eq) andDMAP (0.5 eq). The mixture was heated to 50° C. Upon reaction completion(5 hr), the mixture was cooled to 20° C. and adjusted to pH 8.5 withaqueous NaHCO₃. Methyl tert-butyl ether was added, and the product wasextracted into the aqueous layer. Dichloromethane was added, and themixture was adjusted to pH 3 with aqueous citric acid. Theproduct-containing organic layer was washed with a mixture of pH=3citrate buffer and saturated aqueous sodium chloride. Thisdichloromethane solution of 37 was used without isolation in thepreparation of compound 38.

Preparation of 38: To the solution of compound 37 was addedN-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HONB) (1.02 eq),4-dimethylaminopyridine (DMAP) (0.34 eq), and then1-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (1.1eq). The mixture was heated to 55° C. Upon reaction completion (4-5 hr),the mixture was cooled to 20° C. and washed successively with 1:1 0.2 Mcitric acid/brine and brine. The dichloromethane solution underwentsolvent exchange to acetone and then to N,N-dimethylformamide, and theproduct was isolated by precipitation from acetone/N,N-dimethylformamideinto saturated aqueous sodium chloride. The crude product was reslurriedseveral times in water to remove residual N,N-dimethylformamide andsalts. Yield=70% of 38 from compound 36. Introduction of the activated“Tail” onto the disulfide anchor-resin was performed in NMP by theprocedure used for incorporation of the subunits during solid phasesynthesis.

Preparation of the Solid Support for Synthesis of Morpholino Oligomers:This procedure was performed in a silanized, jacketed peptide vessel(custom made by ChemGlass, NJ, USA) with a coarse porosity (40-60 μm)glass frit, overhead stirrer, and 3-way Teflon stopcock to allow N2 tobubble up through the frit or a vacuum extraction. Temperature controlwas achieved in the reaction vessel by a circulating water bath.

The resin treatment/wash steps in the following procedure consist of twobasic operations: resin fluidization and solvent/solution extraction.For resin fluidization, the stopcock was positioned to allow N2 flow upthrough the frit and the specified resin treatment/wash was added to thereactor and allowed to permeate and completely wet the resin. Mixing wasthen started and the resin slurry mixed for the specified time. Forsolvent/solution extraction, mixing and N2 flow were stopped and thevacuum pump was started and then the stopcock was positioned to allowevacuation of resin treatment/wash to waste. All resin treatment/washvolumes were 15 mL/g of resin unless noted otherwise.

To aminomethylpolystyrene resin (100-200 mesh; ˜1.0 mmol/g N2substitution; 75 g, 1 eq, Polymer Labs, UK, part #1464-X799) in asilanized, jacketed peptide vessel was added 1-methyl-2-pyrrolidinone(NMP; 20 ml/g resin) and the resin was allowed to swell with mixing for1-2 hr. Following evacuation of the swell solvent, the resin was washedwith dichloromethane (2×1-2 min), 5% diisopropylethylamine in 25%isopropanol/dichloromethane (2×3-4 min) and dichloromethane (2×1-2 min).After evacuation of the final wash, the resin was fluidized with asolution of disulfide anchor 34 in 1-methyl-2-pyrrolidinone (0.17 M; 15mL/g resin, ˜2.5 eq) and the resin/reagent mixture was heated at 45° C.for 60 hr. On reaction completion, heating was discontinued and theanchor solution was evacuated and the resin washed with1-methyl-2-pyrrolidinone (4×3-4 min) and dichloromethane (6×1-2 min).The resin was treated with a solution of 10% (v/v) diethyl dicarbonatein dichloromethane (16 mL/g; 2×5-6 min) and then washed withdichloromethane (6×1-2 min). The resin 39 (see FIG. 4) was dried under aN2 stream for 1-3 hr and then under vacuum to constant weight (±2%).Yield: 110-150% of the original resin weight.

Determination of the Loading of Aminomethylpolystyrene-disulfide resin:The loading of the resin (number of potentially available reactivesites) is determined by a spectrometric assay for the number oftriphenylmethyl (trityl) groups per gram of resin.

A known weight of dried resin (25±3 mg) is transferred to a silanized 25ml volumetric flask and ˜5 mL of 2% (v/v) trifluoroacetic acid indichloromethane is added. The contents are mixed by gentle swirling andthen allowed to stand for 30 min. The volume is brought up to 25 mL withadditional 2% (v/v) trifluoroacetic acid in dichloromethane and thecontents thoroughly mixed. Using a positive displacement pipette, analiquot of the trityl-containing solution (500 μL) is transferred to a10 mL volumetric flask and the volume brought up to 10 mL withmethanesulfonic acid.

The trityl cation content in the final solution is measured by UVabsorbance at 431.7 nm and the resin loading calculated in trityl groupsper gram resin (μmol/g) using the appropriate volumes, dilutions,extinction coefficient (ε: 41 μmol-1 cm-1) and resin weight. The assayis performed in triplicate and an average loading calculated.

The resin loading procedure in this example will provide resin with aloading of approximately 500 μmol/g. A loading of 300-400 in μmol/g wasobtained if the disulfide anchor incorporation step is performed for 24hr at room temperature.

Tail loading: Using the same setup and volumes as for the preparation ofaminomethylpolystyrene-disulfide resin, the Tail can be introduced intothe molecule. For the coupling step, a solution of 38 (0.2 M) in NMPcontaining 4-ethylmorpholine (NEM, 0.4 M) was used instead of thedisulfide anchor solution. After 2 hr at 45° C., the resin 39 was washedtwice with 5% diisopropylethylamine in 25% isopropanol/dichloromethaneand once with DCM. To the resin was added a solution of benzoicanhydride (0.4 M) and NEM (0.4 M). After 25 min, the reactor jacket wascooled to room temperature, and the resin washed twice with 5%diisopropylethylamine in 25% isopropanol/dichloromethane and eight timeswith DCM. The resin 40 was filtered and dried under high vacuum. Theloading for resin 40 is defined to be the loading of the originalaminomethylpolystyrene-disulfide resin 39 used in the Tail loading.

Solid Phase Synthesis: Morpholino Oligomers were prepared on a GilsonAMS-422 Automated Peptide Synthesizer in 2 mL Gilson polypropylenereaction columns (Part #3980270). An aluminum block with channels forwater flow was placed around the columns as they sat on the synthesizer.The AMS-422 will alternatively add reagent/wash solutions, hold for aspecified time, and evacuate the columns using vacuum.

For oligomers in the range up to about 25 subunits in length,aminomethylpolystyrene-disulfide resin with loading near 500 μmol/g ofresin is preferred. For larger oligomers,aminomethylpolystyrene-disulfide resin with loading of 300-400 μmol/g ofresin is preferred. If a molecule with 5′-Tail is desired, resin thathas been loaded with Tail is chosen with the same loading guidelines.

The following reagent solutions were prepared:

Detritylation Solution: 10% Cyanoacetic Acid (w/v) in 4:1dichloromethane/acetonitrile; Neutralization Solution: 5%Diisopropylethylamine in 3:1 dichloromethane/isopropanol; CouplingSolution: 0.18 M (or 0.24 M for oligomers having grown longer than 20subunits) activated Morpholino Subunit of the desired base and linkagetype and 0.4 M N ethylmorpholine, in 1,3-dimethylimidazolidinone.Dichloromethane (DCM) was used as a transitional wash separating thedifferent reagent solution washes.

On the synthesizer, with the block set to 42° C., to each columncontaining 30 mg of aminomethylpolystyrene-disulfide resin (or Tailresin) was added 2 mL of 1-methyl-2-pyrrolidinone and allowed to sit atroom temperature for 30 min. After washing with 2 times 2 mL ofdichloromethane, the following synthesis cycle was employed:

Step Volume Delivery Hold time Detritylation 1.5 mL Manifold 15 secondsDetritylation 1.5 mL Manifold 15 seconds Detritylation 1.5 mL Manifold15 seconds Detritylation 1.5 mL Manifold 15 seconds Detritylation 1.5 mLManifold 15 seconds Detritylation 1.5 mL Manifold 15 secondsDetritylation 1.5 mL Manifold 15 seconds DCM 1.5 mL Manifold 30 secondsNeutralization 1.5 mL Manifold 30 seconds Neutralization 1.5 mL Manifold30 seconds Neutralization 1.5 mL Manifold 30 seconds Neutralization 1.5mL Manifold 30 seconds Neutralization 1.5 mL Manifold 30 secondsNeutralization 1.5 mL Manifold 30 seconds DCM 1.5 mL Manifold 30 secondsCoupling 350 uL − 500 uL Syringe 40 minutes DCM 1.5 mL Manifold 30seconds Neutralization 1.5 mL Manifold 30 seconds Neutralization 1.5 mLManifold 30 seconds DCM 1.5 mL Manifold 30 seconds DCM 1.5 mL Manifold30 seconds DCM 1.5 mL Manifold 30 seconds

The sequences of the individual oligomers were programmed into thesynthesizer so that each column receives the proper coupling solution(A,C,G,T,I) in the proper sequence. When the oligomer in a column hadcompleted incorporation of its final subunit, the column was removedfrom the block and a final cycle performed manually with a couplingsolution comprised of 4-methoxytriphenylmethyl chloride (0.32 M in DMI)containing 0.89 M 4-ethylmorpholine.

Cleavage from the resin and removal of bases and backbone protectinggroups: After methoxytritylation, the resin was washed 8 times with 2 mL1-methyl-2-pyrrolidinone. One mL of a cleavage solution consisting of0.1 M 1,4-dithiothreitol (DTT) and 0.73 M triethylamine in1-methyl-2-pyrrolidinone was added, the column capped, and allowed tosit at room temperature for 30 min. After that time, the solution wasdrained into a 12 mL Wheaton vial. The greatly shrunken resin was washedtwice with 300 μL of cleavage solution. To the solution was added 4.0 mLconc aqueous ammonia (stored at −20° C.), the vial capped tightly (withTeflon lined screw cap), and the mixture swirled to mix the solution.The vial was placed in a 45° C. oven for 16-24 hr to effect cleavage ofbase and backbone protecting groups.

Initial Oligomer Isolation: The vialed ammonolysis solution was removedfrom the oven and allowed to cool to room temperature. The solution wasdiluted with 20 mL of 0.28% aqueous ammonia and passed through a 2.5×10cm column containing Macroprep HQ resin (BioRad). A salt gradient (A:0.28% ammonia with B: 1 M sodium chloride in 0.28% ammonia; 0-100% B in60 min) was used to elute the methoxytrityl containing peak. Thecombined fractions were pooled and further processed depending on thedesired product.

Demethoxytritylation of Morpholino Oligomers: The pooled fractions fromthe Macroprep purification were treated with 1 M H3PO4 to lower the pHto 2.5. After initial mixing, the samples sat at room temperature for 4min, at which time they are neutralized to pH 10-11 with 2.8%ammonia/water. The products were purified by solid phase extraction(SPE).

Amberchrome CG-300M (Rohm and Haas; Philadelphia, Pa.) (3 mL) is packedinto 20 mL fritted columns (BioRad Econo-Pac Chromatography Columns(732-1011)) and the resin rinsed with 3 mL of the following: 0.28%NH4OH/80% acetonitrile; 0.5M NaOH/20% ethanol; water; 50 mM H3PO4/80%acetonitrile; water; 0.5 NaOH/20% ethanol; water; 0.28% NH4OH.

The solution from the demethoxytritylation was loaded onto the columnand the resin rinsed three times with 3-6 mL 0.28% aqueous ammonia. AWheaton vial (12 mL) was placed under the column and the product elutedby two washes with 2 mL of 45% acetonitrile in 0.28% aqueous ammonia.The solutions were frozen in dry ice and the vials placed in a freezedryer to produce a fluffy white powder. The samples were dissolved inwater, filtered through a 0.22 micron filter (Pall Life Sciences,Acrodisc 25 mm syringe filter, with a 0.2 micron HT Tuffryn membrane)using a syringe and the Optical Density (OD) was measured on a UVspectrophotometer to determine the OD units of oligomer present, as wellas dispense sample for analysis. The solutions were then placed back inWheaton vials for lyophilization.

Analysis of Morpholino Oligomers: MALDI-TOF mass spectrometry was usedto determine the composition of fractions in purifications as well asprovide evidence for identity (molecular weight) of the oligomers.Samples were run following dilution with solution of3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid),3,4,5-trihydoxyacetophenone (THAP) or alpha-cyano-4-hydoxycinnamic acid(HCCA) as matrices.

Cation exchange (SCX) HPLC was performed using a Dionex ProPac SCX-10,4×250 mm column (Dionex Corporation; Sunnyvale, Calif.) using 25 mM pH=5sodium acetate 25% acetonitrile (Buffer A) and 25 mM pH=5 sodium acetate25% acetonitrile 1.5 M potassium chloride (buffer B) (Gradient 10-100% Bin 15 min) or 25 mM KH2PO4 25% acetonitrile at pH=3.5 (buffer A) and 25mM KH2PO4 25% acetonitrile at pH=3.5 with 1.5 M potassium chloride(buffer B) (Gradient 0-35% B in 15 min). The former system was used forpositively charged oligomers that do not have a peptide attached, whilethe latter was used for peptide conjugates.

Purification of Morpholino Oligomers by Cation Exchange Chromatography:The sample is dissolved in 20 mM sodium acetate, pH=4.5 (buffer A) andapplied to a column of Source 30 cation exchange resin (GE Healthcare)and eluted with a gradient of 0.5 M sodium chloride in 20 mM sodiumacetate and 40% acetonitrile, pH=4.5 (buffer B). The pooled fractionscontaining product are neutralized with conc aqueous ammonia and appliedto an Amberchrome SPE column. The product is eluted, frozen, andlyophilized as above.

Example 8 In Vitro and In Vivo Testing of the Oligomers

A PMO (5′-GCT ATT ACC TTA ACC CAG-3; SEQ ID NO: 1) designed to restorecorrect splicing in the enhanced green fluorescent protein (EGFP) genewas modified at the intersubunit linkage and/or the 5′ and/or 3′ end toproduce modified PMOs, which were evaluated for their splice-correctionactivity and tissue distribution in the EGFP-654 transgenic mouse model(Sazani, Gemignani et al. 2002). In this model, the EGFP-654 geneencoding for functional EGFP is interrupted by an aberrantly-splicedmutated intron, and cellular uptake of EGFP targeted PMOs, such as SEQID NO: 1, can be evaluated by RT-PCR detection of the restored EGFP-654splice product in tissues or detection of functional EGFP byfluorescence.

Sequences of the oligomers employed for the experiments described hereinare presented in Table 2. The oligomers typically comprise a piperazinelinker on the 5′ end and either an ethylene glycol trimer (EG3) orguanidinyl terminal group linked thereto. The 3′ end is eitherunmodified (H) or comprises a trityl, gaunidinyl or peptide moiety. An“*” indicates the presence of a modified intersubunit linkage, thestructure of which is provided in Table 3. All other linkages are PMO.

TABLE 2 Oligomer Sequences Number Sequence Modifications 3′ End 5′ End0-1-0-730 GCT ATT ACC TTA ACC CAG (SEQ ID NO: 2) None H EG3 NG-10-0389GC*T AT*T ACC T*TA ACC CAG (SEQ ID NO: 3) * = G-pip Trityl GuanidineNG-12-0127 GC*T A*T*T ACC T*TA ACC CAG (SEQ ID NO: 4) * = map H EG3NG-12-0128 GC*T A*T*T ACC *T*TA ACC CAG (SEQ ID NO: 5) * = map H EG3NG-12-0153 *G*C*T *A*T*T *A*C*C *T*T*A *A*C*C *C*A*G * = morph H EG3(SEQ ID NO: 6) NG-12-0157 GC*T A*T*T ACC T*TA ACC CAG (SEQ ID NO: 7)* = MG-pip C═(NH)NH₂ EG3 NG-12-0158GC*T A*T*T ACC *T*TA ACC CAG (SEQ ID NO: 8) * = MG-pip C═(NH)NH₂ EG3NG-08-0524 GCT ATT ACC TTA ACC CAG (SEQ ID NO: 9) 3′-peptide(RXRRBR)₂-XB EG3 NG-11-0153 GCT ATT ACC TTA ACC CAG (SEQ ID NO: 10)3′-peptide R₆-G EG3 NG-10-0110GC*T AT*T ACC T*TA ACC CAG (SEQ ID NO: 11) * = guanidinyl Guanidinyl EG3NG-10-0299 GC*T AT*T ACC T*TA ACC CAG (SEQ ID NO: 12) * = apn H EG3

TABLE 3 Structure of Intersubunit Linkages Name Structure G-pip

Map

MG-pip

Apn

Morph

PMO

Tissue Distribution

EGFP-654 transgenic mice (14-28 weeks old, female) were injected by tailvein I.V. with a G-pip modified oligomer (NG-10-0389) or apeptide-modified oligomer (NG-08-0524, X=aminohexanoic acid,B=beta-alanine) at doses of 2.5 mg/kg, 5 mg/kg, 10 mg/kg and 20 mg/kg(PBS vehicle). Tissues were harvested 7 days post injection and analyzedfor presence of the oligomers. FIG. 1 presents the tissue distributiondata for the 10 mg/kg dose. The data show that the G-pip modifiedoligomer has a higher affinity for T-cells relative to the peptidemodified oligomer.

In Vitro Delivery to Lymphocytes

Splenocytes from an EGFP-654 reporter mouse were harvested and a cellsuspension was made. Cells were CD3/CD28 stimulated in the presence ofthe oligomers from Table 2 for 24 h at 37° C. Cells were harvested fromthe plate and run through a flow cytometer for detection of EGFP and Tand B cell markers. Activated and resting T cells were delineated usingCD25 as the activation marker. Successful delivery to T cells isindicated by the presence of EGFP within the cell (i.e., EGFP positivecells).

FIGS. 2-6 show the frequency of EGFP positive cells within eachlymphocyte population for the oligomers from Table 2. FIGS. 2 and 3present data for activated T cells and B cells, respectively. The dataindicate that intersubunit linkages containing guanidinyl,alkylguanidinyl or alkylaminyl groups have higher efficacy in T cellsand B cells relative to unmodified PMO (dimethylamine linkages) andmorpholino PMO (morpholino linkages).

FIGS. 4, 5 and 6 present data for activated T cells, resting T cells andB cells, respectively. This data shows that oligomers having guanidinylmodifications in the intersubunit linkages having higher efficacy in Tand B cells relative to peptide conjugated oligomers, especially atlower concentrations.

In Vivo Delivery to Lymphocytes

EGFP-654 reporter mice were injected with various oligomers from Table 2as described above. Splenocytes were harvested 7 days-post injection andstained for T regulatory markers and EGFP fluorescence. The frequency ofEGFP positive cells in the oligomer treated cohort was divided by thefrequency of EGFP positive cells in saline treated mice to generate thefold increase in EGFP over vehicle. Results are presented in FIGS. 7-10.Again, the data indicate that intersubunit linkages containingguanidinyl, alkylguanidinyl or alkylaminyl groups have higher efficacyin T cells and macrophages relative to unmodified PMO (dimethylaminelinkages) and other types of PMO modifications (e.g. peptide).

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, including butnot limited to U.S. Provisional Patent Application No. 62/171,102 filedon Jun. 4, 2015, are incorporated herein by reference, in theirentirety. Aspects of the embodiments can be modified, if necessary toemploy concepts of the various patents, applications and publications toprovide yet further embodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1. A method for treatment of a lymphocyte-related disease or condition,the method comprising administering an effective amount of an oligomerto a patient in need thereof, wherein the oligomer comprises a backbonehaving a sequence of morpholino ring structures joined by intersubunitlinkages, the intersubunit linkages joining a 3′-end of one morpholinoring structure to a 5′-end of an adjacent morpholino ring structure,wherein each morpholino ring structure is bound to a base-pairingmoiety, such that the oligomer can bind in a sequence-specific manner toa target nucleic acid, wherein at least one of the intersubunit linkageshas the following structure (I):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein: R¹ is guanidinyl, alkylguanidinyl or alkylaminyl; L¹is absent or present, and when present is selected from alkylene,aminoalkylene, oxyalkylene and thioalkylene; X is, at each occurrence,independently S or O; Y is, at each occurrence, independently —O— or—NH—; and Z is an optionally substituted 5, 6 or 7-membered heterocyclicring.
 2. The method of claim 1, wherein the morpholino ring structureshave the following structure (i):

wherein B is, at each occurrence, independently a base-pairing moiety.3. The method of any one of claims 1-2, wherein Z is an optionallysubstituted 5 or 6-membered heterocyclic ring.
 4. The method of claim 3,wherein Z is pyrrolidinyl, or piperidinyl.
 5. The method of claim 4,wherein Z is piperidinyl.
 6. The method of claim 4, wherein Z has one ofthe following structures:


7. The method of claim 6, wherein Z has the following structure:


8. The method of claim 7, wherein Z has the following structure:


9. The method of any one of claims 1-8, wherein R¹ is guanidinyl. 10.The method of any one of claims 1-8, wherein R¹ is alkylguanidinyl. 11.The method of claim 10, wherein alkylguanidinyl has the followingstructure:

wherein R′ is C₁-C₆alkyl.
 12. The method of claim 11, wherein R′ ismethyl.
 13. The method of any one of claims 1-8, wherein R¹ isalkylaminyl.
 14. The method of claim 13, wherein alkylaminyl is —NHR″,where R″ is C₁-C₆alkyl.
 15. The method of claim 14, wherein R″ ismethyl.
 16. The method of any one of claim 1-15, wherein L¹ is absent.17. The method of any one of claims 1-16, wherein X is O.
 18. The methodof any one of claims 1-17, wherein Y is —O—.
 19. The method of any oneof claims 1-13, wherein at least one of the intersubunit linkages hasthe following structure (II):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein: R² and R³ are each independently H or C₁-C₆alkyl, X′ is S or O;and Y′ is —O— or —NH—.
 20. The method of claim 19, wherein R² and R³ areeach methyl.
 21. The method of claim 19 or 20 wherein X′ is O.
 22. Themethod of any one of claims 19-21, wherein Y′ is —O—.
 23. The method ofclaim 1, wherein at least one of the intersubunit linkages has thefollowing structure:


24. The method of claim 1, wherein at least one of the intersubunitlinkages has the following structure:


25. The method of claim 1, wherein at least one of the intersubunitlinkages has the following structure:


26. The method of any one of claims 1-25, wherein the lymphocyte-relateddisease or condition is a T-cell-related disease or condition.
 27. Themethod of claim 26, wherein the T-cell is an activated T-cell.
 28. Themethod of claim 26, wherein the T-cell is a CD4 or CD8 cell.
 29. Themethod of any one of claims 1-28, wherein the disease or condition iscancer or an autoimmune disease or condition.
 30. A method for treatmentof a T-cell related disease or condition, the method comprisingcontacting activated T-cells with an oligomer comprising a backbonehaving a sequence of morpholino ring structures joined by intersubunitlinkages, the intersubunit linkages joining a 3′-end of one morpholinoring structure to a 5′-end of an adjacent morpholino ring structure,wherein each morpholino ring structure is bound to a base-pairingmoiety, such that the oligomer can bind in a sequence-specific manner toa target nucleic acid, wherein at least one of the intersubunit linkageshas the following structure (I):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein: R¹ is guanidinyl, alkylguanidinyl or alkylaminyl; L¹is absent or present, and when present is selected from alkylene,aminoalkylene, oxyalkylene, oxoalkylene and thioalkylene; X is, at eachoccurrence, independently S or O; Y is, at each occurrence,independently —O— or —NH—; and Z is an optionally substituted 5, 6 or7-membered heterocyclic ring.
 31. Use of an oligomer comprising abackbone having a sequence of morpholino ring structures joined byintersubunit linkages, the intersubunit linkages joining a 3′-end of onemorpholino ring structure to a 5′-end of an adjacent morpholino ringstructure, wherein each morpholino ring structure is bound to abase-pairing moiety, such that the oligomer can bind in asequence-specific manner to a target nucleic acid, wherein at least oneof the intersubunit linkages has the following structure (I):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein: R¹ is guanidinyl, alkylguanidinyl or alkylaminyl; L¹ is absentor present, and when present is selected from alkylene, aminoalkylene,oxyalkylene, oxoalkylene and thioalkylene; X is, at each occurrence,independently S or O; Y is, at each occurrence, independently —O— or—NH—; and Z is an optionally substituted 5, 6 or 7-membered heterocyclicring, for preparation of a pharmaceutical composition for treatment of alymphocyte-related disease or condition.